Compositions and uses of antimicrobial materials with tissue-compatible properties

ABSTRACT

Compositions comprising a mixture of an antimicrobial cationic polypeptide and a second pharmaceutically-acceptable polymer are disclosed, as well as methods and uses thereof for the treatment and prevention of infections that occur when our natural barriers of defense are broken.

INCORPORATION BY REFERENCE TO ANY PRIORITY APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/240,197, filed on Aug. 18, 2016, which is a continuation of U.S.patent application Ser. No. 14/385,752, filed on Sep. 16, 2014, which isthe U.S. National Phase entry of International ApplicationPCT/US2013/032535, filed on Mar. 15, 2013, which claims the benefit ofpriority to U.S. Provisional Application No. 61/615,150, filed on Mar.23, 2012, U.S. Provisional Application No. 61/625,757, filed on Apr. 18,2012, U.S. Provisional Application No. 61/625,760, filed on Apr. 18,2012, and U.S. Provisional Application No. 61/716,242, filed on Oct. 19,2012, the disclosures of each of which are hereby incorporated byreference in their entireties for all purposes.

REFERENCE TO SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitledAMIC001C1SeqListing.TXT, which was created and last modified on Sep. 15,2020 and is 44,633 bytes in size. The information in the electronicSequence Listing is hereby incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

When barriers are broken, infections occur. Surgery, trauma and burns,medical instrumentation (e.g., catheterization, ventilation), chronicwounds (e.g., diabetic foot ulcers) and a variety of diseases disruptour natural barriers of defense. In nearly all cases, these “wounds”become contaminated with microbes. Then, the terrain of wounds providesan excellent environment for microbial growth. The battle begins, andit's trench warfare. Damaged tissues (‘cracks and crevices”), alteredblood flow and exudate production, changes in local temperature, pH andtissue oxygenation, as well as the lack of commensal bacteria, can allcontribute. Also, bleeding and vascular leakage may provide fluids andnutrients that ultimately support microbial growth. Bacterial or fungalcolonization, and/or overt infection may occur. Microbial biofilms canhelp microbes to create their own local environments. Various surgical,trauma and medical settings all involve disruption of our naturalbarriers of defense and deserve special attention because the outcomescan range from rapid cure to lethal sepsis.

Natural barriers are typically referred to by their anatomical sitessuch as skin, pulmonary epithelium, gastrointestinal mucosa, etc. Thesenames may imply a level of simplicity that is unwarranted. Thesebarriers are often both passive and active. They can involve a varietyof cells, secreted glycoproteins, matrix components and fluids that actin concert to provide effective defense against microbial invasion. Insome sites, resident microbes contribute to the barrier action againstother potential invaders. Under most circumstances, these physical andfunctional barriers are highly effective. However, they can be brokenrather easily by mechanical or chemical insults. In addition, certainsystemic diseases can weaken our natural barriers and increase the riskof breakdown, as occurs in diabetic foot ulcers or cystic fibrosis.Finally, a first infection can weaken host defenses against a secondinfection, as occurs in influenza followed by bacterial pneumonia orTrichomonas vaginalis followed by certain sexually transmitted diseases(e.g., HIV).

Broken barriers of defense leave the host susceptible to infection by awide variety of microbes, ranging from typically benign commensalorganisms to aggressive pathogens. Commonly, we are our own source ofthe microbes that contaminate our wounds. The human body hosts a verylarge number of bacteria, predominately on skin, in mouths and withinlower GI tracts. It has been estimated that there are more bacterialcells (10¹⁴) than mammalian cells (10¹³) within the space of one humanbody. Despite this close relationship with microbes, most of our tissues(including blood, subcutaneous tissue, muscle, brain) remain sterileuntil the disruption of the natural barriers. Other people andenvironmental sources of microbes are also important, especially inhealthcare settings. Once a barrier is broken, microbial contamination,critical level colonization, biofilm formation and/or overt infectionmay occur. Polymicrobial colonization and/or infection is common incertain settings (e.g., diabetic foot ulcers, complex intra-abdominalinfections), and may involve aerobes, anaerobes or both.

Prior approaches to the prevention and treatment of these infectionshave demonstrated substantial weaknesses. Both lack of effectiveness andtissue toxicity have been challenges. Antimicrobials often fail to getto the right tissue spaces and/or fail to remain active for sufficienttime to prevent or treat infection. Complex surfaces like those of theabdominal cavity or large burns are particularly difficult to covereffectively. Finally, safe application of sufficient antimicrobialmaterials into certain tissue spaces (such as through laparoscopic orarthroscopic equipment) can be challenging. Antimicrobials that arereadily applied by these methods tend to be solution-based materialswith limited ability to bind tissues and remain active over time.

Biofilms present a particular challenge. Increasing evidence points tothe resistance of bacterial biofilms to a variety of antimicrobialapproaches and to their role in adverse patient outcomes. Thesemicrobial communities resist traditional antiseptics and antibioticsthrough several mechanisms, including, but not limited to, their ownproduction of extracellular polymeric substances, which are oftennegatively charged (anionic). Penetration of these materials bytraditional antimicrobials is often limited. For example, in acutewounds (e.g. surgery and trauma), devitalized tissue and foreign bodies(e.g., prosthetic implants) may support biofilm formation and therebyincrease the probability of overt infection. In chronic wounds (e.g.diabetic foot ulcers), biofilms may persist and lead to delayed woundhealing. Medical instruments like ventilators and catheters can be asite of biofilm formation and provide a source of infection.

Antimicrobial treatment of early infections may alter the course of theinfection, resulting in more resistant and more dangerous infections.Common antimicrobial strategies focus on the use of selectiveantibiotics (e.g., penicillin for gram-positive organisms) in order toavoid the development of bacteria that are resistant to broad-spectrumantibiotics. Inadvertently, this important strategy can have negativeoutcomes on an individual patient, where targeted antibiotics result inthe emergence of an aggressive, different microorganism (e.g.,Pseudomonas). In this way, treated wounds can become the site for a“parade of pathogens”, where an early, dominant microbial species (e.g.,Staph aureus) is replaced by a second (e.g., MRSA, methicillin-resistantStaph aureus) and, perhaps, even a third and fourth microbial species(e.g., a multi-drug resistant gram negative species).

The large numbers of adverse patient outcomes in today's advancedhealthcare settings underscore the inadequacies of prior art in theprevention and treatment of these infections. Several key weaknessesinclude:

1. Low antimicrobial activity in tissue settings and on biofilms;

2. Inadequate distribution to the relevant tissue space;

3. Limited, if any, barrier activity;

4. Narrow breadth of antimicrobial activity enables the “parade ofpathogens”;

5. Inadequate treatment fosters more antimicrobial resistance; and/or

6. Tissue toxicity.

Infections of wounds and other broken-barrier settings are common andcostly. In the US alone, approximately 12 million traumatic injuries aretreated in emergency departments each year. In addition, there are morethan 50 million surgeries (inpatient and outpatient). The US Departmentof Health and Human Services indicates that there are more than 1.7million healthcare-associated infections annually, resulting inapproximately 100,000 deaths and $30 billion in healthcare costs peryear. Many of these healthcare-associated infections start with brokenbarriers. Examples include surgical site infections (SSIs),catheter-associated urinary tract infections and ventilator-associatedpneumonia. The chronic wounds associated with pressure ulcers (bedsores) and diabetic foot ulcers present their own unique challenges.

In addition to infection, several other wound-associated outcomes remainmajor challenges. These include blood loss, tissue adhesions/scarring,and poor wound healing. And, in some cases, known antimicrobialtreatments make these problems worse. Certain antimicrobial woundtreatments (including antibiotic washes) can result in excessive tissueresponses (e.g., tissue adhesions or scarring). Certainantiseptic/antimicrobial materials may alter wound healing, resulting ininsufficient tissue responses (e.g., poor wound healing, poor woundstrength).

Effective hemostasis in wounds also remains a substantial problem.Hemostatic materials have been described and are utilized in a varietyof settings, including in trauma and in surgery. While effective in somesituations, these materials do not provide ideal solutions to thechallenges. First, there are times when the hemostasis is insufficientand too much bleeding occurs, potentially with lethal consequences. Insome of these cases initial hemostasis occurs, however, subsequentre-bleeding occurs. This may be due to fibrinolytic activity. Inaddition to the problems resulting from blood loss, extravasated bloodcomponents in the tissues may contribute to additional adverse outcomesincluding infection and the fibrotic responses seen with post-surgicaltissue adhesions. Second, in some cases, hemostatic materials causeproblems by entering the blood stream and causing clotting (thrombosis)within blood vessels, potentially with lethal outcomes. Third, in somecases, wound treatment materials (including hemostatic materials) canserve as a site for subsequent infection or can result in abnormaltissue responses such as adhesion formation and/or tissue scarring,resulting in adverse medical outcomes. Improved approaches to hemostasisare needed.

SUMMARY OF THE INVENTION

In accordance with embodiments of the invention, a mixture of anantimicrobial cationic polypeptide and a secondpharmaceutically-acceptable polymer is used in the treatment andprevention of infections that occur when our natural barriers of defenseare broken. These novel compositions can provide two functions: directantimicrobial activity and barrier activity. Embodiments of theinvention addresses one or more weaknesses of previous antimicrobialsdescribed above. Notably, it has been recognized that the effectivenessof most of the previous antiseptics and antibiotics were based ondeterminations of antimicrobial activity in solution with the microbesin suspension (MIC assays), producing results that are not necessarilyindicative of effectiveness in tissues, and thus could lead away fromdetermining effectiveness in tissues. By contrast, the inventors focusedon the design and selection of agents for broad antimicrobial activity,especially at tissue surfaces and in the terrain (“cracks and crevices”)of wounds. This includes the formation of a barrier containing cationic(positively charged) elements that can inhibit the movement of certainsubstances or cells that display anionic elements (e.g., microbes). Inone embodiment, the synthetic cationic polypeptides of thesecompositions contain at least one cationic segment and at least onehydrophobic segment, are comprised substantially of natural amino acids,and are broadly antimicrobial (i.e., against gram positive and gramnegative bacteria). They can also be designed to self-assemble, based inpart on the interaction of their hydrophobic segments. Further,synthetic polypeptides are formulated with a secondpharmaceutically-acceptable polymer to provide a composition that isdirectly antimicrobial and that effectively coats tissues. Thesemixtures may also display hemostatic properties. In some embodiments,the second pharmaceutically acceptable polymer is not a polyethyleneglycol (PEG).

Embodiments of the invention may be used alone or in combination withother materials that provide similar or complementary activities.

An embodiment provides aqueous composition for the prevention,inhibition, or treatment of infection comprising: a mixture comprisingone or more synthetic, cationic polypeptide(s) with antimicrobialactivity; and a second pharmaceutically-acceptable polymer that is not asynthetic, cationic polypeptide(s) with antimicrobial activity; whereinthe amounts of the one or more synthetic, cationic polypeptide(s) andthe second pharmaceutically-acceptable polymer are each at least about100 μg/mL based on the total volume of the aqueous composition; whereinthe amount of the second pharmaceutically-acceptable polymer is at leastabout 10% by weight, based on the weight of the one or more synthetic,cationic polypeptide(s); and wherein the synthetic, cationicpolypeptide(s) and the second pharmaceutically-acceptable polymer aremutually miscible in water.

The synthetic, cationic polypeptide(s) with antimicrobial activity andthe second pharmaceutically-acceptable polymer are considered mutuallymiscible if at least about 90% of the polymeric components remainmutually soluble 24 hours after mixing and maintaining at roomtemperature in water at a concentration of each polymer of 1 mg/mL, uponvisible examination.

In another embodiment, one or more of the synthetic cationicpolypeptide(s) in the aqueous composition comprises a segment having achain length of at least 40 amino acid residues.

In another embodiment, the synthetic cationic polypeptide(s) in theaqueous composition comprises substantially all natural amino acidsubunits.

In another embodiment, the synthetic cationic polypeptide(s) in theaqueous composition is characterized by at least one segment containingat least five consecutive cationic amino acid residues and at least onesegment containing at least five consecutive hydrophobic amino acidresidues.

In another embodiment, the second pharmaceutically-acceptable polymer inthe aqueous composition is selected from the group consisting ofcellulose, alginate, collagen, polymeric surfactant, polyethyleneglycol, polyvinyl alcohol, polyurethane, polyvinyl pyrolidinone (PVP),fibrin(ogen), blood proteins and tissue proteins.

In another embodiment, the antimicrobial activity of the aqueouscomposition is greater than 3 logs killing of Staphylococcus epidermidisand Escherichia coli in standard 60 minute time-kill assays at asynthetic cationic polypeptide(s) concentration of 100 μg/mL or less.

In another embodiment, the aqueous composition is further characterizedby the ability to disrupt or inhibit a biofilm in vitro at a totalpolymer concentration of 40 mg/ml or less.

In another embodiment, the aqueous composition is further characterizedby a barrier activity, as measured by a decrease in the diffusion rateof an anionic dye of more than 2 logs at a total polymer concentrationof 40 mg/mL or less.

In another embodiment, the aqueous composition is further characterizedby a storage modulus of at least 50 Pa at a total polymer concentrationof less than 40 mg/mL.

In another embodiment, the aqueous composition is further characterizedby a storage modulus of at least 50 Pa at a total polymer concentrationof less than 40 mg/mL and an ability to pass through a 20 g needle usingless than 60 N pressure.

In another embodiment, the aqueous composition is further characterizedby an ability to pass through a 20 g needle and recover a minimum of 70%of its strength as measured by storage modulus within 10 minutes.

In another embodiment, the aqueous composition is in the form of asolution, a gel, a cream, a foam, or a dressing.

In another embodiment, the aqueous composition is further characterizedas being in combination with, or binding to, a dressing material,including but not limited to a gauze or sponge.

In another embodiment, the aqueous composition has pro-coagulantactivity, pro-hemostatic activity, or both.

In another embodiment, the aqueous composition further comprises anactive pharmaceutical ingredient (API) selected from the groupconsisting of steroid, pro-inflammatory agent, anti-inflammatory agent,anti-acne agent, preservatives hemostatic agent, angiogenic agent, woundhealing agent, anti-cancer agent and other antimicrobial agent.

Another embodiment provides a use of any one of the aqueous compositionsdescribed herein for any one or more selected from the group consistingof prevention of infections, treatment of infections, treatment fortopical anti-infection, treatment for microbial decolonization, woundtreatment, surgical site treatment, trauma treatment, burn treatment,treatment of diabetic foot ulcers, eye treatment, treatment of vaginalinfections, treatment of urinary tract infections, hand sanitization,for coating prosthetic devices and/or implants, food preservation andsolution preservation.

Another embodiment provides a method for the prevention and/or treatmentof infections comprising: contacting a tissue of a patient subject withany of the aqueous compositions described herein.

In another embodiment, the method further comprises applyingnegative-pressure to a wound.

In another embodiment, the method further comprises treating the patientsystemically with other antibiotics and/or locally with anotherantimicrobial, and/or at least one selected from the group consisting ofan antibiotic, an anti-biofilm agent, a surfactant, and a combinationthereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B depict examples of synthetic cationic polypeptides,K_(x)(rac-L)_(y) and K_(x)L_(y), respectively. These polypeptidescontain a cationic segment of lysine amino acids and a hydrophobicsegment of leucine amino acids. The cationic segments provide formultimeric interaction with anionic substances, including bacterialsurfaces. The hydrophobic segments associate in aqueous media, leadingto the formation of various structures, such as multimers in solution,micelles, sheets, and fibrils. These hydrophobic interactions areimportant in barrier formation.

FIGS. 2A and 2B are tables that show examples of synthetic cationicpolypeptides synthesized using either (A) an amine (shown in FIG. 2A) or(B) an organometallic Co(PMe₃)₄ initiator (shown in FIG. 2B).

FIG. 3 is a gel permeation chromatogram of a chain extension experiment.

FIG. 4 is a gel permeation chromatogram of a representative deprotecteddiblock copolypeptide.

FIGS. 5A and 5B show attenuated total reflectance fourier-transforminfrared (ATR-IR) interferograms of Leucine N-carboxy anhydride (NCA)and TFA-Lysine NCA monomers, respectively.

FIGS. 6A and 6B show attenuated total reflectance fourier-transforminfrared (ATR-IR) interferograms of a crude reaction mixture and acompleted polymerization, respectively.

FIGS. 7A and 7B show a matrix-assisted laser desorption ionization(MALDI) mass spectrum of a representative peptide and an expanded viewof a portion of that MALDI spectrum, respectively.

FIG. 8 shows a liquid proton nuclear magnetic resonance (H¹—NMR)spectrum of a representative deprotected copolypeptide in d-TFA.

FIG. 9 shows an in vitro antimicrobial time-kill assay (60 min) againstS. epidermidis (RMA 18291), and E. coli (ATCC 25922) using K₁₀₀(rac-L)₂₀(SEQ ID NO: 12).

FIG. 10 shows an in vitro antimicrobial time-kill (60 min) against S.epidermidis (RMA 18291), and E. coli (ATCC 25922) using K₁₀₀L₄₀ (SEQ IDNO: 19).

FIGS. 11A and 11B show in vitro antimicrobial time-kills ofK₁₀₀(rac-L)₂₀ (SEQ ID NO: 12) and 1:1 K₁₀₀(rac-L)₂₀ (SEQ ID NO:12):Poloxamer 407 solutions, respectively, at concentrations of 10 and100 μg/mL against S. aureus (29213), and P. aeruginosa (27853) after 5and 60 min.

FIG. 12 is a table (Table 3) that shows an in vitro antimicrobialtime-kill of K₁₀₀(rac-L)₂₀ (SEQ ID NO: 12) solutions at concentrationsof 10 and 100 μg/mL against a variety of gram positive and gram negativebacteria and fungi after 60 min contact time.

FIG. 13 shows that K₁₀₀(rac-L)₂₀ (SEQ ID NO: 12) and K₁₀₀L₄₀ (SEQ ID NO:19) at concentrations of 0.1-20 mg/mL are effective against P.aeruginosa biofilms in vitro (24 hour contact time).

FIGS. 14A and 14B show in vitro antimicrobial time-kill assays againstS. aureus (29213), and P. aeruginosa (27853), respectively, after 5 and60 min, using K₁₀₀(rac-L)₂₀ (SEQ ID NO: 12) at concentrations of 10 and100 μg/mL with other ratios of excipients: 5:1 K₁₀₀(rac-L)₂₀ (SEQ ID NO:12):Poloxamer 407, 2:1 K₁₀₀(rac-L)₂₀ (SEQ ID NO: 12):HEC, and 1:3K₁₀₀(rac-L)₂₀ (SEQ ID NO: 12):Peg 400.

FIG. 15 shows an in vitro antimicrobial time-kill assay against S.aureus (29213) after 30 min, using K₁₀₀L₄₀ (SEQ ID NO: 19) (10 μg/mL)alone and in combination with cellulose ethers (methyl cellulose (MC),hydroxypropylmethyl cellulose (HPMC), hydroxypropyl cellulose (HPC), andhydroxyethyl cellulose (HEC)) at a 1:1 ratio.

FIG. 16 is a table (Table 4) that shows an in vitro antimicrobial timekill assay against S. aureus (29213), S. epidermidis (RMA 18291), P.aeruginosa (27853), and E. coli (25922) after 60 min. contact time.K₁₀₀L₄₀ (SEQ ID NO: 19) at concentrations of 10 and 100 μg/mL with otherratios of hydroxyethyl cellulose: 1:2 K₁₀₀(rac-L)₂₀ (SEQ ID NO: 12):HECand 1:20 K₁₀₀(rac-L)₂₀ (SEQ ID NO: 12) were tested.

FIGS. 17A and 17B show barrier assays using K₁₀₀L₄₀ (SEQ ID NO: 19) andpolyethylene glycol (PEG), respectively. FIG. 17A shows that a 2%synthetic cationic polypeptide preparation (K₁₀₀L₄₀ (SEQ ID NO: 19)) washighly effective in blocking the diffusion of a colored, anionic dyeover a 48 hour period. By contrast, as shown in FIG. 17B, the dyediffused readily (within 5 minutes) through a 2% PEG (10,000)preparation.

FIG. 18 shows a synthetic cationic polypeptide binding to Vitroskin©, asynthetic tissue analogue. A substantial portion of applied FITC-labeledcopolypeptides were shown to remain associated with Vitroskin©, asdemonstrated using 1% solutions of FITC-K₁₀₀(rac-L)₂₀ (SEQ ID NO: 12)and FITC-K₁₀₀L₄₀ (SEQ ID NO: 19). By comparison, the majority of labeledBSA was removed by washing. The % remaining was determined using FITCfluorescence (λ_(exc)=495 nm, λ_(em)=521 nm) after 1-10 washes andcalculated as 100% minus the percentage removed.

FIG. 19 shows in vitro antimicrobial activity of K₁₀₀(rac-L)₂₀ (SEQ IDNO: 12) and K₁₀₀L₄₀ (SEQ ID NO: 19) (100-3000 μg/mL) on a Vitroskin©surface against S. aureus after 0 and 5 washes (5×1 mL). Each 3 cm×3 cmpiece of Vitroskin© was incubated with S. aureus for 60 min.

FIGS. 20A and 20B show A) firmness values for mixtures of 1% solutionsof synthetic cationic polypeptides with 1% hydroxyethyl cellulose (HEC,Natrosol HHX) in water (FIG. 20A); and B) work of adhesion values formixtures of 1% solutions of copolypeptides with 1% hydroxyethylcellulose (HEC, Natrosol HHX) in water (FIG. 20B).

FIG. 21 is a table (Table 5) that shows synthetic cationic polypeptidetexture analysis profile data of pure polypeptides and HEC mixtures. a.Values for 1% (w/w) polypeptide in water. b. Values for 1%polypeptide/1% HEC (w/w) in water. c. Interaction parameter:ΔF=F_(mix(poly/HEC))−(F_(poly)+F_(HEC)). WhereF_(mix(poly/HEC))=firmness of 1% polypeptide/1% HEC, F_(poly)=firmnessof 1% polypeptide solution, and F_(HEC)=firmness of 1% HEC (NatrosolHHX) in water. Similar treatment of data was performed for the work ofadhesion interaction parameter.

FIGS. 22A and 22B show A) Strain and B) frequency sweep of synergisticmixture of 1% K₁₀₀L₄₀ (SEQ ID NO: 19)/1% HEC and individual componentsat 1% in water, respectively. ▪=1% K₁₀₀L₄₀ (SEQ ID NO: 19)/1% HEC, ●=1%K₁₀₀L₄₀ (SEQ ID NO: 19), and ▾=1% HEC. G′ values=filled symbols andG″=open symbols.

FIG. 23 demonstrates that synthetic cationic polypeptide solutions(anti-infective solutions; left) and synthetic cationic polypeptidehydrogels (barrier gel; right) are effective at coating open wounds in aporcine model. Synthetic cationic polypeptides werefluorescently-labeled.

FIG. 24 shows the antimicrobial activity of K₁₀₀(rac-L)₂₀ (SEQ ID NO:12) in a rodent infection model. Polypropylene mesh was insertedsubcutaneously in rats followed by 10⁷ MRSA (33593). After 15 min.,either 10, 2 or 0.4 mg/mL of K₁₀₀(rac-L)₂₀ (SEQ ID NO: 12) or water wasadded. After 2 days, implanted mesh and surrounding tissue were analyzedfor MRSA bacterial counts.

FIG. 25 shows the antimicrobial activity of K₁₀₀(rac-L)₂₀ (SEQ ID NO:12) in an open-wound porcine model. Each wound was contaminated withbacteria (S. epidermidis, P. aeruginosa (pig clinical isolate)). After 2hrs, wounds were rinsed with saline and treated with 5 mL of testarticle or water. Test articles: K₁₀₀(rac-L)₂₀ (SEQ ID NO: 12) at 10, 2,0.4 mg/mL, 10 mg/mL K₁₀₀(rac-L)₂₀ (SEQ ID NO: 12) and 30 mg/mL PEG 400in water, and deionized water as a control. Gauze soaked in test articleor water was placed on top of the wounds. Wounds were biopsied at 4 hrsafter treatment.

FIG. 26 shows that a K₁₀₀L₄₀ (SEQ ID NO: 19) hydrogel prevents infectionin an open-wound porcine model. To each wound hydrogel was applied tothe wound bed and to gauze. The hydrogel-soaked gauze was placed on topof wounds. Saline was used as a control. After 15 min. each wound wascontaminated with bacteria (S. epidermidis, P. aeruginosa (pig clinicalisolate)). Hydrogel test articles: K₁₀₀L₄₀ (SEQ ID NO: 19) 10 mg/mL,K₁₀₀L₄₀ (SEQ ID NO: 19) 5 mg/mL and 10 mg/mL HEC, K₁₀₀L₄₀ (SEQ ID NO:19) 2 mg/mL and 15 mg/mL HEC, and saline as a control. Wounds werebiopsied at 4 hrs after hydrogel application.

FIG. 27 depicts synthetic cationic polypeptides bound to amedically-acceptable sponge material. This product concept illustratesone way that copolypeptides could be brought into contact with wounds inorder to facilitate the delivery of hemostatic and/or antimicrobialactivity.

FIG. 28 shows the results of an in vitro whole blood clotting assay withK₁₀₀L₄₀ (SEQ ID NO: 19) and a 1:1 mixture of K₁₀₀L₄₀ (SEQ ID NO: 19) andHEC at 10 and 100 μg/mL. Controls were HEC alone at 10 and 100 μg/mL anda negative control of saline and a positive control of thromboplastin,TF (50 μL in 500 μL of whole blood).

FIG. 29 shows the results of a platelet aggregation assay with K₁₀₀L₄₀(SEQ ID NO: 19) and a 1:1 mixture of K₁₀₀L₄₀ (SEQ ID NO: 19) and HEC at10 and 100 μg/mL. Controls were HEC alone at 10 and 100m/mL, a negativecontrol of saline, and a positive control of collagen.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

In accordance with embodiments of the invention, an aqueous compositionthat includes a mixture of an antimicrobial cationic polypeptide and asecond pharmaceutically-acceptable polymer is used in the treatmentand/or prevention of infections that occur when our natural barriers ofdefense are broken. This novel composition can display two functions:direct antimicrobial activity and barrier activity. Embodiments of theinvention addresses one or more weaknesses of previous antimicrobialsdescribed above. Notably, the inventors began with the recognition thatmost of the previous antiseptics and antibiotics were based onantimicrobial activity in solution with the microbes in suspension (MICassays), a method that could lead away from effectiveness in tissues. Bycontrast, the inventors focused on the design and selection of agentsfor broad antimicrobial activity, especially at tissue surfaces and inthe terrain (“cracks and crevices”) of wounds. This includes theformation of a barrier containing cationic (positively charged) elementsthat can inhibit the movement of certain substances or cells thatdisplay anionic elements (e.g., microbes). In one embodiment, thesynthetic cationic polypeptides of these compositions contain at leastone cationic segment and at least one hydrophobic segment, are comprisedsubstantially of natural amino acids, and are broadly antimicrobial(i.e., against gram positive and gram negative bacteria). They can alsobe designed to self-assemble, based in part on the interaction of theirhydrophobic segments. Further, synthetic polypeptides are formulatedwith a second pharmaceutically-accepted polymer to provide a compositionthat is directly antimicrobial and that effectively coats tissues. Thesemixtures may also display hemostatic properties. In some embodiments,the second pharmaceutically acceptable polymer is not a polyethyleneglycol (PEG).

Embodiments of the invention may be used alone or in combination withother materials that provide similar or complementary activities.

Synthetic cationic polypeptides. A variety of synthetic cationicpolypeptides can be used in the aqueous compositions described herein.In an embodiment, the synthetic cationic polypeptide comprises a segmentof recurring units or residues of positively charged amino acids (e.g.lysine, arginine) and another segment of recurring units or residues ofhydrophobic amino acids (e.g. leucine, isoleucine, valine, alanine).Examples include copolypeptides composed of a segment or block of one ormore recurring lysine amino acids and a segment or block of one or morerecurring leucine amino acids with the overall structure of K_(x)L_(y)(FIGS. 1A and 1B). In various embodiments, one or more of the syntheticcationic polypeptide(s) comprise a segment having a chain length of atleast 40 amino acid residues. In various embodiments, the syntheticcationic polypeptide(s) comprises substantially all natural amino acidsubunits. In some embodiments, the synthetic cationic polypeptide(s) ischaracterized by at least one segment containing at least fiveconsecutive cationic amino acid residues and at least one segmentcontaining at least five consecutive hydrophobic amino acid residues.For example, in some cases, the polypeptides include one or moresegments comprising at least 5 consecutive recurring lysine amino acidunits and one or more segments comprising at least 5 consecutivehydrophobic recurring amino acids (e.g., leucine). In some cases,cationic polypeptides may be relatively long-chain molecules havinglysine blocks containing 50 to 200 or more lysine amino acid residues.Examples of synthetic cationic polypeptides include K₅₀(rac-L)₁₀ (SEQ IDNO: 1), K₅₀(rac-L)₂₀ (SEQ ID NO: 2), K₅₀(rac-L)₃₀ (SEQ ID NO: 3),K₅₀(rac-L)₄₀ (SEQ ID NO: 4), K₅₀(rac-L)₅₀ (SEQ ID NO: 5), K₅₀L₁₀ (SEQ IDNO: 6), K₅₀L₂₀ (SEQ ID NO: 7), K₅₀L₃₀ (SEQ ID NO: 8), K₅₀L₄₀ (SEQ ID NO:9), K₅₀L₅₀ (SEQ ID NO: 10), K₁₀₀(rac-L)₁₀ (SEQ ID NO: 11), K₁₀₀(rac-L)₂₀(SEQ ID NO: 12), K₁₀₀(rac-L)₃₀ (SEQ ID NO: 13), K₁₀₀(rac-L)₄₀ (SEQ IDNO: 14), K₁₀₀(rac-L)₅₀ (SEQ ID NO: 15), K₁₀₀L₁₀ (SEQ ID NO: 16), K₁₀₀L₂₀(SEQ ID NO: 17), K₁₀₀L₃₀ (SEQ ID NO: 18), K₁₀₀L₄₀ (SEQ ID NO: 19),K₁₀₀L₅₀ (SEQ ID NO: 20), K₂₀₀(rac-L)₁₀ (SEQ ID NO: 21), K₂₀₀(rac-L)₂₀(SEQ ID NO: 22), K₂₀₀(rac-L)₃₀ (SEQ ID NO: 23), K₂₀₀(rac-L)₄₀ (SEQ IDNO: 24), K₂₀₀(rac-L)₅₀ (SEQ ID NO: 25), K₂₀₀L₁₀ (SEQ ID NO: 26),K₂₀₀L₂₀₂₀ (SEQ ID NO: 27), K₂₀₀L₃₀ (SEQ ID NO: 28), K₂₀₀L₄₀ (SEQ ID NO:29), K₂₀₀L₅₀ (SEQ ID NO: 30) (FIGS. 2A and 2B). Other synthetic cationicpolypeptides (e.g. with longer or shorter lysine segments and longer orshorter leucine segments) are also envisioned. Also, this inventionincludes other synthetic cationic polypeptides where the cationicsegments can include other amino acids, such as arginine, andhydrophobic blocks that can contain one or more hydrophobic amino acids,including but not limited to leucine, alanine, valine, and/orisoleucine. In some cases, one or more of the segments may have a randomsequence of amino acids, including hydrophobic and hydrophilic aminoacids.

Examples of synthetic cationic polypeptides include those described inU.S. Pat. Nos. 6,680,365; 6,632,922; 6,686,446; 6,818,732; 7,329,727; USPublished Patent Application No. 2008/0125581; and US Published PatentApplication No. 2011/048869. The aforementioned patents and patentapplications are hereby incorporated herein by reference, andparticularly for the purpose of describing synthetic cationicpolypeptides and method of making them.

Methods of manufacturing synthetic cationic polypeptides that haveexceptionally narrow polydispersities with high reproducibility havebeen developed, and such polymers can be made to various specificationsto suit the needs of the antimicrobial activity and the barrierformation. For example, by combining high quality a-amino acid N-carboxyanhydrides (NCAs) in anhydrous solvents with a benzyl amine initiator, ahigh quality block copolypeptide is synthesized. These polymers thenundergo deprotection and purification to yield the final product.

Several analytical techniques have been developed and refined to bothmonitor the synthesis of the peptides and to analyze the resultingpolymeric products for size, properties and residual impurities.Infrared spectroscopy may be used to monitor the progress of thereaction, while Size Exclusion Gel Permeation Chromatography may be usedto monitor the growth and status of the polymer at various stages of theprocess. Other analytical techniques such as nuclear magnetic resonancespectroscopy (NMR), matrix assisted laser desorption ionization massspectroscopy (MALDI-MS), and inductively coupled plasma massspectroscopy (ICP-MS) may be used, e.g., as additional quality controltests to ensure consistent reproducibility and purity (FIG. 3-8 ).

Synthetic cationic polypeptides can be designed to demonstratebroad-based antimicrobial activity (see US Published Patent ApplicationNo. 2011/048869). A synthetic cationic polypeptide is considered to haveantimicrobial activity if it provides greater than 3 logs killing ofStaphylococcus epidermidis and Escherichia coli in a standard 60 minutetime-kill assays at a synthetic cationic polypeptide concentration of1.0 mg/mL or less. In an embodiment, the synthetic cationic polypeptidehas an antimicrobial activity that provides greater than 3 logs killingof Staphylococcus epidermidis and Escherichia coli in a standard 60minute time-kill assays at a synthetic cationic polypeptideconcentration of 100 μg/mL or less. As shown in FIGS. 9-12 , embodimentsof these synthetic cationic polypeptides demonstrate antimicrobialactivity in vitro against both Gram-positive and Gram-negative bacteria.This activity is demonstrated in in vitro time-kill assays with thesynthetic cationic polypeptides in aqueous media. Further, embodimentsof synthetic cationic polypeptides demonstrated anti-biofilm activity invitro, as depicted in FIG. 13 . It is likely that these effects can beexplained in part by the cationic polypeptides binding to the anioniccharges present in the matrix of the biofilm. In addition, thesurfactant-like activity of the synthetic cationic polypeptides maycontribute to the anti-biofilm effect. It is also recognized that theinventive compositions may change the structure and/or charge ofbiofilms, allowing other agents (e.g., locally applied or systemicallyapplied antibiotics) to penetrate the biofilm, thereby enhancingactivity.

Mixtures of synthetic cationic polypeptides and other polymers canretain antimicrobial activity in vitro. The secondpharmaceutically-acceptable polymer is different from the one or moresynthetic, cationic polypeptide(s) having antimicrobial activity. In anembodiment, the second pharmaceutically-acceptable polymer has little orno antimicrobial activity itself. For example, in an embodiment, theantimicrobial activity of second pharmaceutically-acceptable polymer isless than 10% of the antimicrobial activity of the synthetic, cationicpolypeptide(s).

The individual amounts of the synthetic, cationic polypeptide(s) and thesecond pharmaceutically-acceptable polymer in the aqueous compositionare at least about 100 μg/mL, and can be higher, e.g. about 1 mg/mL,about 5 mg/mL, or about 10 mg/mL, or about 20/ml, or about 40 mg/ml orhigher. The amount of the second pharmaceutically-acceptable polymer inthe aqueous composition is at least about 10% by weight, based on theweight of the one or more synthetic, cationic polypeptide(s), and may behigher, e.g., at least about 20% by weight, at least about 30% byweight, or at least about 50% by weight, same basis. The synthetic,cationic polypeptide(s) and the second pharmaceutically-acceptablepolymer in the aqueous composition are selected such the polymers aremutually miscible. As noted above, the synthetic, cationicpolypeptide(s) with antimicrobial activity and the secondpharmaceutically-acceptable polymer are considered mutually miscible ifat least about 90% of the polymeric components remain mutually soluble24 hours after mixing and maintaining at room temperature in water at aconcentration of each polymer of 1 mg/mL, upon visible examination.Surprisingly, such mutual miscibility of the water, synthetic, cationicpolypeptide(s) and the second pharmaceutically-acceptable polymer can beachieved, despite the expectation of phase separation due to the typicalmutual incompatibility of polymers in aqueous solution at the 1 mg/mLconcentrations and molecular weights described herein. The aqueouscompositions described herein can be prepared by intermixing theindividual polymeric components with water, e.g., at room temperaturewith stirring, using ordinary mixing methods known to those skilled inthe art.

Example 1

As depicted in FIGS. 14A-16 , embodiments of synthetic cationicpolypeptides were shown to retain substantially all of theirantimicrobial activity in the presence of otherpharmaceutically-acceptable polymers, as demonstrated by in vitrotime-kill assays against S. aureus, S. epidermidis, P. aeruginosa, andE. coli. In these studies, a variety of cellulose-based second polymerswere evaluated. As shown in FIGS. 11 and 13 , other studies demonstratedretained antimicrobial activity in in vitro the presence of otherpharmaceutically-acceptable (polymeric surfactants). In an embodiment,the antimicrobial activity of the aqueous composition is greater than 3logs killing of Staphylococcus epidermidis and Escherichia coli instandard 60 minute time-kill assays at a synthetic cationicpolypeptide(s) concentration of 100 μg/mL or less. In anotherembodiment, the aqueous composition is further characterized by theability to disrupt or inhibit a biofilm in vitro at a total polymerconcentration of 40 mg/ml or less.

Mixtures of synthetic cationic polypeptides and other polymers canexhibit enhanced physical and viscoelastic properties in vitro. Indeveloping compositions for use in patients, the need to maintainantimicrobial activity while enhancing volume, tissue coverage areas,and/or biocompatibility has been recognized. Various synthetic cationicpolypeptides have been combined with other pharmaceutically-acceptablepolymers in a way to retain at least about 80% (e.g., at least 90%) ofthe antimicrobial activity of the synthetic cationic polypeptides and,in preferred embodiments, also enhance tissue coverage. It is believedthat the amphiphilic nature of the synthetic cationic polypeptides andtheir self-assembly in aqueous solution induce stable phase-separated(collapsed or solvated) dispersions of hydrophobic and hydratedhydrophilic material. In some embodiments, it is believed that the chainlength of the copolypeptides (e.g., degree of polymerization or n>50)provides a network of partially collapsed pockets of hydrophobicity thatslow down solute and solvent diffusion. The barrier nature of thematerials is a product of the ability of the materials to trap and slowdown water mobility, and by consequence dramatically slow down thepassage of any solute or particle present in water. The length of thepolypeptide strongly influences the barrier properties (e.g., bydrastically reduced diffusion). Therefore, selection of a second polymerfor mixing with the synthetic cationic peptide(s) should be undertakencarefully to avoid disruption of influential biophysical parameters andto achieve mutual solubility in the aqueous composition. Those skilledin the art can use routine experimentation guided by the teachingsprovided herein to select the polymeric components and amounts to formthe aqueous compositions described herein.

The properties of the aqueous compositions described herein can be tunedby controlling the ratio of the amount of the cationic polypeptide tothe second pharmaceutically-acceptable polymer(s). Non-limiting examplesof these second polymers may include celluloses (e.g,hydroxyethylcellulose (HEC), hydroxypropylcellulose (HPC),hydroxymethylcellulose (HMC)), alginates, collagens, polymericsurfactants, polyethylene glycols, polyvinyl alcohols, polyurethanes,polyvinyl pyrolidinones (PVP), fibrin(ogen), or blood or tissueproteins. In some embodiments, the second pharmaceutically acceptablepolymer is not a polyethylene glycol (PEG). It is believed that thebarrier of the composition is in dynamic equilibrium with its individualcomponents, at least one of which is also antimicrobial. The leaching ofaggregates may be an advantage because the shed material may increasethe effective surface area of the barrier which may increase theeffectiveness of the interaction between microbes and the barriermaterial (FIG. 17A-19 ). Barrier properties of the materials can befurther enhanced by the synergistic effect of polar biopolymers thatfurther crosslink the self-assembled co-polypeptide matrix. Byformulating with different biopolymers the flow and mechanicalcharacteristics of the materials, as well as the strength of the barriercan be adjusted and controlled.

Example 2

A texture analysis profile has been used to determine the effects ofblock copolypeptide composition and hydrophobic enantiopurity on themechanical properties of the block copolypeptide/HEC mixtures (FIGS. 20Aand 20B). Measurement of firmness values for individual componentsindicated that a 1% (w/w) solution of K₁₀₀L₄₀ (SEQ ID NO: 19) in waterhad a firmness of 2.94+/−0.25 mN and a solution of 1% (w/w) HEC in waterdemonstrated a firmness of 5.38+/−0.32 mN (FIG. 21 (Table 5)). Mixing 1%(w/w) K₁₀₀L₄₀ (SEQ ID NO: 19) and 1% HEC (w/w) together resulted in asubstantial increase in firmness, to a value of 22.77+/−0.90 mN. Thiscorresponds to an interaction parameter value of 14.45 mN and an overallincrease of over 170% over what would be expected from the additivecontributions of the individual components. As shown in FIG. 20 b , asimilar trend was observed for adhesiveness. By comparison, thecombination of the diblock copolypeptide K₁₀₀(rac-L)₄₀ (SEQ ID NO: 14)(containing a racemic hydrophobic block) at 1% (w/w) and HEC at 1% (w/w)concentration also demonstrated enhanced firmness and adhesiveness;however, the increase was not as pronounced as with K₁₀₀L₄₀ (SEQ ID NO:19), containing an enantiopure hydrophobic block. Hydrophobic blocklength was also shown to be influential. Mixtures of 1% (w/w) K₁₀₀L₂₀(SEQ ID NO: 17) and 1% (w/w) HEC showed increased firmness over eitherbiopolymer alone, but the effect was smaller than that of K₁₀₀L₄₀ (SEQID NO: 19). Interestingly, K₁₀₀L₂₀ (SEQ ID NO: 17) was antagonistic forwork of adhesion. Further, K₁₀₀(rac-L)₂₀ (SEQ ID NO: 12), with itsshorter racemic hydrophobic block, showed no effect. The lysinehomopolypeptides, K₁₀₀ (SEQ ID NO: 31) and K₂₀₀ (SEQ ID NO: 32), alsofailed to enhance the firmness or adhesiveness of HEC alone, and infact, appeared to demonstrate antagonistic activity.

Example 3

Rheological measurements further support the synergistic interactionsbetween K₁₀₀L₄₀ (SEQ ID NO: 19) and HEC. In FIGS. 22A and 22B, effectsare seen in both the oscillatory strain sweep and frequency dependentsweep. By itself, 1% (w/w) K₁₀₀L₄₀ (SEQ ID NO: 19) showedcharacteristics of a relatively brittle, weak gel. In strain sweepanalysis, the brittle nature of the gel was observed by the breakdown ofthe gel network at low strain rates around γ=0.01; and frequency sweepanalysis indicated the formation of a weak gel with an elastic modulus(G′=22 Pa at 1 rad/s). By comparison, 1% (w/w) HEC showed rheologicalproperties more characteristic of a viscous fluid. Strain sweep analysisof a 1% HEC solution demonstrated a higher loss modulus (G″) thanelastic modulus (G′) throughout all strain rates tested (FIG. 22A) andthe frequency sweep also showed higher G″ over G′ values until higherfrequencies (ca. 100 rad/s) were reached (FIG. 22B). Substantial changesin rheological properties were observed upon mixing 1% (w/w) K₁₀₀L₄₀(SEQ ID NO: 19) with 1% (w/w) HEC. Notably, strain sweep analysis of themixture showed an extension of the linear viscoelastic region by anorder of magnitude showing a decrease in G′ around γ=0.1. The frequencysweep showed a large, synergistic increase in the elastic modulus G′=141Pa at 1 rad/s, which is a seven-fold increase over the elastic modulusof 1% (w/w) K₁₀₀L₄₀ (SEQ ID NO: 19) alone (G′=22 Pa).

The amounts and types of polymeric components in the aqueouscompositions described herein can be selected to achieve variousproperties. In an embodiment, the aqueous composition is characterizedby a barrier activity, as measured by a decrease in the diffusion rateof an anionic dye of more than 2 logs at a total polymer concentrationof 40 mg/mL or less. In an embodiment, the aqueous composition ischaracterized by a storage modulus of at least 50 Pa at a total polymerconcentration of less than 40 mg/mL. In an embodiment, the aqueouscomposition is characterized by a storage modulus of at least 50 Pa at atotal polymer concentration of less than 40 mg/mL and an ability to passthrough a 20 g needle using less than 60 N pressure. In an embodiment,the aqueous composition is characterized by an ability to pass through a20 g needle and recover a minimum of 70% of its strength as measured bystorage modulus within 10 minutes. Those skilled in the art can useroutine experimentation guided by the teachings provided herein toselect the polymeric components and amounts to form aqueous compositionshaving the properties described herein.

Example 4

Mixtures of synthetic cationic polypeptides and other polymers canexhibit enhanced antimicrobial activity in vivo. Notably, syntheticcationic polypeptides, as micellar solutions and as hydrogels, can beused to coat tissues in vivo (FIG. 23 ). This tissue coating may involvethe binding of the cationic peptides, through charge interactions, toanionic charges displayed on damaged tissues (as well as anionic chargesin bacterial biofilms). In addition, self-assembly and cross-linking mayincrease the amount of material that binds and coats a tissue (e.g.thinner or thicker coating) and therefore influence a variety ofbiological activities and responses.

As depicted in FIG. 24 , these synthetic cationic polypeptides areantimicrobial when locally applied in vivo. In seeking to developimproved products for in vivo applications, it has been recognized thattissue binding and tissue coating properties of these materials maysubstantially affect their activities, including their antimicrobialproperties, their anti-biofilm properties, and their effects on tissueadhesion formation and tissue remodeling. Molecular characteristics(e.g. length, charge, etc.) of the synthetic cationic polypeptides, aswell as structures that they form in aqueous environments (e.g.micelles, sheets, fibrils, hydrogels) may affect tissue binding andtissue coating. It has also been recognized that mixing these syntheticcationic polypeptides with other polymers to form aqueous compositionsas described herein can alter, in various ways, their biophysicalproperties in aqueous media and on tissues. Therefore, we testedmixtures of synthetic cationic polypeptides and other polymers in vivo.

Example 5

Aqueous compositions that include mixtures of synthetic cationicpolypeptides with two different polymers (polyethylene glycol 400 andhydroxyethyl cellulose) were both found to be effective in vivo. Asdepicted in FIG. 25 , K₁₀₀(rac-L)₂₀ (SEQ ID NO: 12) was effective in anporcine open-wound treatment model alone and in combination with PEG400. As depicted in FIG. 26 , K₁₀₀L₄₀ (SEQ ID NO: 19) was effective atpreventing microbial contamination alone and in combination withhydroxyethyl cellulose. Further, the data suggests that enhancedbiophysical properties of the mixtures can improve antimicrobialactivity in vivo over the synthetic cationic copolypeptides alone. Theaqueous compositions described herein can be used for any one or moretreatments and/or applications, including but not limited to preventionof infections, treatment of infections, treatment for topicalanti-infection, treatment for microbial decolonization, wound treatment,surgical site treatment, trauma treatment, burn treatment, treatment ofdiabetic foot ulcers, eye treatment, treatment of vaginal infections,treatment of urinary tract infections, hand sanitization, for coatingprosthetic devices and/or implants, food preservation and solutionpreservation.

The aqueous compositions described herein may be formulated assolutions, emulsions, particles, or hydrogels with a variety ofviscoelastic properties to enhance their antimicrobial properties, theirbarrier properties, or both. In one embodiment, an aqueous compositionas described herein comprises a wound wash product with a singlelysine-leucine block copolypeptide in water, saline, or other aqueousmedia that is mixed with a second polymer, typically a surfactant suchas poloxamer 407. In one embodiment, an aqueous composition as describedherein may comprise a viscous fluid/flowing gel that can be appliedthrough a sprayer to coat various tissues. This could be used in open orlaparoscopic approaches. These materials may by themselves or incombination with other materials be formed into a variety of dressingsor bandages. These may include constituting or coating a variety ofmaterials such as gauze or sponges. An example would include an aqueouscomposition as described herein (e.g., containing a synthetic blockcopolypeptide KxLy) in the form of a coating on gauze or alginatebandages. Another example would include a two-layer material where anaqueous composition as described herein (e.g., containing a syntheticblock copolypeptide KxLy alone or with another polymer such as acollagen) coats a face of a relatively inert sponge material (e.g.polyacrylate, polyurethane, or polyhema) (FIG. 27 ). In this case theembodiment could have the appearance of a rectangular sponge with onecoated surface or of a spherical sponge where the entire surface iscoated (reminiscent of a tennis ball). This may be particularlyadvantageous in the treatment of bleeding wounds where hemostasis isrequired (FIG. 28-29 ).

An embodiment provides a method for the prevention and/or treatment ofinfections that includes contacting a tissue of a subject with anaqueous composition as described herein, e.g., to a wound. Anotherembodiment further includes applying negative-pressure to the treatedwound. The subject can be an animal, preferably a human. In anembodiment, the subject is further treated systemically with anantibiotic and/or locally with another antimicrobial, and/or at leastone selected from the group consisting of an antibiotic, an anti-biofilmagent, a surfactant, and a combination thereof.

The aqueous compositions described herein can further include one ormore of an active pharmaceutical ingredient (API). Examples of such APIsinclude steroids, pro-inflammatory agents, anti-inflammatory agents,anti-acne agents, preservatives, hemostatic agents, angiogenic agents,wound healing agents, anti-cancer agents and other antimicrobial agents.

What is claimed is:
 1. A method for prevention, inhibition, or treatmentof infection, comprising: selecting a site capable of supporting biofilmformation; applying to the site a composition comprising one or moresynthetic polypeptide(s) having a length of at least 40 amino acidresidues; wherein the one or more synthetic polypeptide(s) has a netcationic charge at neutral pH; wherein the one or more syntheticpolypeptide(s) inhibits or kills microbes in biofilms.